Original Article Diallyl Trisulfide Inhibits Proliferation and Promotes Apoptosis of Side Population Cells in Multiple Myeloma Cells

Int J Clin Exp Med 2017;10(3):5749-5756 www.ijcem.com /ISSN:1940-5901/IJCEM0038165

Original Article Diallyl trisulfide inhibits proliferation and promotes apoptosis of side population cells in multiple myeloma cells

Junquan Zeng1, Tingting Liu2, Yongliang Zheng1, Bin Liu1, Mushui Fang1, Shengping Long1, Yijian Chen3

1Department of Hematology, The Affiliated Hospital of Jinggangshan University, Ji’an 343000, Jiangxi Province, China; 2Department of Hematology, Jiangxi Provincial People’s Hospital, Nanchang 330006, Jiangxi Province, China; 3Department of Hematology, The First Affiliated Hospital of Gannan Medical University, Ganzhou 341000, Jiangxi Province, China Received August 18, 2016; Accepted October 24, 2016; Epub March 15, 2017; Published March 30, 2017

Abstract: Multiple myeloma (MM) is an incurable cancer, in which the main factors leading to death are relapse and the development of resistance to drugs. Both of these are associated with the existence of side population (SP) cells, which have features similar to those of tumor stem cells. The development of new safe agents, possibly from natural products, is necessary to more efficiently treat SP cells involved in MM. Diallyl trisulfide (DATS), a natural organosulfur compound isolated from garlic, has been shown to inhibit the occurrence and development of tumors and cancer stem cells and enhance chemosensitivity. However, the effect of DATS on MM proliferation and the biological behavior of SP cells remain unclear. Therefore, in this study, human MM RPMI-8226 and NCI-H929 cell lines were treated with different concentrations of DATS (2, 5, 10, 20, 40, or 80 μM) for 1, 2, or 3 days and the half-maximal inhibitory concentration (IC50) was evaluated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. In addition, SP RPMI-8226 and NCI-H929 cells were isolated and analyzed using flow cytometry. The effects of dimethyl sulfoxide (DMSO), DATS, and bortezomib on the percentage of MM SP cells, survival rate, colony formation, cell cycle, and cell apoptosis were analyzed using an MTT assay, a colony assay, and flow cytometry analysis. DATS displayed a dose- and time-dependent effect on the survival rate of RPMI-8226 and NCI-H929 cells. DATS and bortezomib treatment reduced the percentage of MM SP cells and the SP cell survival rate and inhibited colony formation and the cell cycle. In addition, DATS and bortezomib treatment promoted SP cell apoptosis. In conclusion, DATS inhibited the proliferation and promoted the apoptosis of SP MM cells.

Keywords: Multiple myeloma, diallyl trisulfide, bortezomib, side population cells

Introduction and drug resistance are associated with the existence of tumor stem cells. Side population Multiple myeloma (MM), a type of cancer that (SP) cells have features similar to those of affects the plasma cells in the bone marrow, cancer stem cells and have been identified in is the second most common hematological MM using flow cytometry along with Hoechst- cancer worldwide [1]. The number of new MM 33342 staining [4-6]. In MM, SP cells have cases is 6.3 per 100000 persons per year and important pathophysiological and clinical impli- the mortality rate is 3.3 deaths per 100000 cations. Therefore, inhibiting the proliferation persons per year [2]. Currently, MM is treated and promoting the apoptosis of SP cells could with chemotherapy and radiation, which have reduce drug resistance and cancer recurrence, improved the survival rate of patients with thereby prolonging the tumor-free interval in MM. However, the disease remains incurable patients and improving survival rates [7-10]. and has a 5-year survival rate of approxima- Bortezomib, a proteasome inhibitor, reduced tely 47% [3]. Tumor relapse and drug resistan- SP cell numbers by suppressing proliferation ce are the main factors leading to death in through inhibition of the cell cycle. However, patients with MM. In addition, tumor relapse the drug has obvious toxic and side effects DATS inhibits proliferation of side population cells in MM cells

[11]. Therefore, the development of new safer Chromadex, Irvine, CA, USA) and incubated in a agents, possibly from natural products, is nec- 96-well plate in a final volume of 0.1 ml at 37°C essary for the efficient treatment of SP cells in for 0, 24, 48, and 72 h. Thereafter, 10 μl of MTT MM. reagent was added to each well and the plate was incubated for 4 h at 37°C. The absorbance Diallyl trisulfide (DATS), a natural organosulfur was measured using a Vmax microplate spec- compound isolated from garlic, has been widely trophotometer (Molecular Devices, Sunnyvale, used in China for more than a thousand years CA, USA) at 570 nm. Each sample was assayed [12]. Recorded history attests that garlic was in triplicate. used to stimulate the immune system, prevent or treat various diseases, and promote health Groups and treatment [13, 14]. In numerous cancer types, DATS inhibited the occurrence and development of tumors SP cells were treated with four drugs: DATS, by inhibiting cell proliferation, migration, and bortezomib (10 nm/L, Selleck Chemicals LLC, invasion, arresting the cell cycle, and promoting Houston, TX, USA) as a positive control, and apoptosis [15, 16]. DATS also inhibited cancer 0.1% dimethyl sulfoxide (DMSO) as a negative stem cells and enhanced chemosensitivity [17, control. Cells were harvested after treatment 18]. However, the effect of DATS on MM prolif- following each assay. eration and the biological behavior of SP cells are still unclear. Hoechst33342 fluorescent staining

Therefore, in this study, MM cells were treated SP cells were sorted from the MM RPMI-8226 with DATS to determine its effect on the surviv- and NCI-H929 cell lines using the Hoechst- al rate of MM cells. In addition, SP cells were 33342 fluorescent staining method with flow sorted from MM cells and treated with DATS to cytometry. The RPMI-8226 and NCI-H929 cells determine the effect of DATS on the biological were seeded into six-well plates, cultured for behavior of SP cells. The results indicated that 48 h, and 10 μg/ml Hoechst33342 stain (Sig- DATS inhibited the survival of MM cells and ma-Aldrich, St. Louis, MO, USA) was added. The decreased the survival rate, inhibited colony cells were further cultured for 20 min. After formation, arrested the cell cycle, and promot- the culture medium containing Hoechst33342 ed the apoptosis of SP cells. was removed, the plates were washed with the medium thrice and sorted using flow cy- Materials and methods tometry.

Cell culture Cell cycle analysis

Human MM Roswell Park Memorial Institute For the cell cycle analysis, each group of cells (RPMI)-8226 and NCI-H929 cell lines were was harvested, digested with trypsin, washed obtained from the American Type Culture Col- twice with PBS, and then centrifuged at 2000 lection (ATCC, Manassas, VA, USA) and cul- rpm for 5 min to collect the cells. Thereafter, tured in RPMI 1640 medium supplemented the cells were fixed with 70% precooled etha- with 10% (v/v) fetal bovine serum (FBS), 100 U/ nol at 4°C overnight, digested with 200 μg/ml ml penicillin, 100 µg/ml streptomycin, and 2 ribonuclease A at 37°C for 30 min, then 100 μl mmol/l L-glutamine (Gibco, Carlsbad, CA, USA). propidium iodide (PI) was added at 4°C in the All cells were maintained in a humidified incu- dark for 30 min. The samples were analyzed bator in a 5% CO2 atmosphere at 37°C. using flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA) and the data were analyzed Cell viability assay using ModFit software.

Cell viability was detected using a 3-(4,5-dimeth- Colony formation assay ylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay according to the manufacturer’s A colony formation assay was performed as protocol (Promega, Madison, WI, USA). Briefly, previously described [19]. Briefly, 2×104 RPMI- RPMI-8226 and NCI-H929 cells (5×103 cells/ 8226 and NCI-H929 cell suspensions were ml) were treated with various concentrations of resuspended in 0.8 ml growth medium con- DATS (Grade: reagent grade (RG); purity 98%; taining 0.3% low-melting-temperature agarose

5750 Int J Clin Exp Med 2017;10(3):5749-5756 DATS inhibits proliferation of side population cells in MM cells

Figure 1. DATS reduced the survival rate of NCI-H929 and RPMI-8226 cells.

(Promega, Madison, WI, USA) and treated with one-way analysis of variance (ANOVA) followed the drugs according to their grouping. The cells by a post-hoc LSD test. An independent t-test were then incubated in 24-well plates in tripli- was used to compare the differences between cate over a base layer of 0.8 ml growth medium groups. P-values <0.05 were considered statis- containing 0.6% low-melting-temperature aga- tically significant. rose. The plates were incubated for two weeks until colonies formed. The colonies were visual- Results ized and their number calculated using lightmi- croscopy (10×). The colony-forming efficiency DATS inhibited MM cell survival (CFE) percentage was calculated using the following formula: The effect of DATS on the survival rate of MM cells was evaluated using an MTT assay. CFE = colony number ÷ inoculation cell number Treatment of RPMI-8226 and NCI-H929 cells ×100% for 1 to 3 days with 2, 5, 10, 20, 40, or 80 μM of DATS reduced the cell survival rate in a Apoptosis assay dose- and time-dependent manner (Figure 1). The IC values of DATS were 67.56, 44.77, and Apoptosis was detected using an apoptosis 50 detection kit (KeyGene, Nanjing, China). RPMI- 23.35 mg/L in NCI-H929 cells at 24, 48, and 72 h, respectively. The IC values of DATS were 8226 and NCI-H929 cells were collected after 50 centrifugation at 2000 rpm for 5 min. The pre- 47.38, 36.54, and 17.92 mg/L in RPMI-8226 cipitates were digested with trypsin and wash- cells at 24, 48, and 72 h, respectively. A DATS ed twice with PBS. The cells were then centri- concentration of 10 mg/L (approximate IC25) fuged at 2000 rpm for 5 min and 5×105 cells was used for subsequent experiments. were suspended in 500 μl of binding buffer from the detection kit. Thereafter, 5 μl each Proportion of SP cells in MM cell lines of Annexin V-fluorescein isothiocyanate (FITC) and PI were added and the cells were incubat- SP cells are known to promote tumor relapse ed for 15 min in the dark. The cells were then and drug resistance in MM [20]. In this study, analyzed immediately using flow cytometry. SP cells were analyzed and isolated using flow cytometry and were detected among RPMI- Statistical analysis 8226 and NCI-H929 cells at a proportion of 2.08±0.438% and 1.26±0.352%, respectively All statistical analyses were performed using (Figure 2). the statistical package for the social sciences (SPSS) version 19.0 software (IBM Inc., USA). DATS reduced the percentage of MM SP cells Continuous variables are presented as mean ± standard deviation (SD). The differences be- To understand the effect of DATS on the sur- tween multiple groups were analyzed using a vival rate of SP cells, the cells were analyzed

5751 Int J Clin Exp Med 2017;10(3):5749-5756 DATS inhibits proliferation of side population cells in MM cells

Figure 2. Proportion of SP cells in MM cell lines. A: Representative images of SP cells detected. B: Percentage of SP cells and main population cells. Data are presented as mean ± SD.

Figure 3. DATS reduced the percentage of MM SP cells. A: Representative image of SP cells detected among NCI- H929 cells. B: Percentage of SP cells among NCI-H929 cells; data are mean ± SD, *P<0.05 vs. DMSO. C: Represen- tative image of SP cells detected among RPMI-8226 cells. D: Percentage of SP cells among RPMI-8226 cells; data are presented as mean ± SD, *P<0.05 vs. DMSO. using flow cytometry after drug treatment Fig-( DATS reduced the SP cell survival rate and in- ure 3). The results indicated that the percent- hibited colony formation and the cell cycle age of SP cells more significantly decreas-ed after treatment with DATS and bortezomib than To understand the effect of DATS on SP cells, it did after treatment with dimethyl sulfoxi- SP cells were separated from MM cells using de (DMSO) (P<0.05). The effect of bortezomib flow cytometry. Survival rate, cell cycle, and treatment was not significantly different from colony formation were evaluated after treat- that of DATS treatment (P>0.05). ment with DMSO, DATS, and bortezomib. The

5752 Int J Clin Exp Med 2017;10(3):5749-5756 DATS inhibits proliferation of side population cells in MM cells

Figure 4. DATS reduced the survival rate and inhibited the colony formation and cell cycle of MM SP cells. A: DATS reduced the survival rate of MM SP cells. B: DATS reduced the colony formation of MM SP cells. C: DATS inhibited the cell cycle of MM SP cells. Bars indicate the cell cycle of MM SP cells in NCI-H929 and RPIM-8216 cells. Data are presented as mean ± SD, *P<0.05 vs. DMSO.

MTT assay indicated that the survival rate of DATS and bortezomib treatment than it did the SP cells more significantly decreased after after DMSO treatment (P<0.05). Furthermore,

5753 Int J Clin Exp Med 2017;10(3):5749-5756 DATS inhibits proliferation of side population cells in MM cells

Figure 5. DATS promoted the apoptosis of MM SP cells. A: DATS promoted the apoptosis of SP cells in NCI-H929 cells. B: DATS promoted the apoptosis of SP cells in RPMI-8226 cells. Bars indicate the apoptosis rate of MM SP cells. Data are presented as mean ± SD, *P<0.05 vs. DMSO. this effect was found to be dose- and time- Discussion dependent in RPMI-8226 and NCI-H929 cells (Figure 4A). In addition, DATS and bortezomib DATS is a natural drug widely used in China to treatment inhibited colony formation more stimulate the immune system and promote significantly than DMSO treatment P ( <0.05) health [12, 13]. Previous studies indicated that (Figure 4B). The cell cycle was analyzed using treatment with DATS suppressed the prolifera- flow cytometry Figure ( 4C). Compared to DM- tion of human breast cancer cells by inhibiting SO treatment, DATS and bortezomib treatment estrogen receptor-α activity [21]. Furthermore, significantly increased the percentage of cells treatment with DATS inhibited the proliferation and induced the apoptosis of bladder cancer in the G1-phase, which prevented their transi- cells by mediating a caspase-dependent signal- tion from the G1- to the S-phase (P<0.05). The ing pathway and regulating the phosphoinosit- results also indicated that there was no sig- ide3-kinase (PI3K)/Akt and JNK pathways [22]. nificant difference between bortezomib treat- In this study, we found that treatment with ment and DATS treatment in regards to ef- DATS reduced the survival rate of MM cells in a fects on survival rate, cell cycle, and colony dose- and time-dependent manner. The results formation (P>0.05). indicated that DATS may be useful as a new agent for the treatment of MM. DATS promoted SP cell apoptosis MM remains incurable, with a 5-year survival SP cell apoptosis was analyzed using flow cy- rate of approximately 47%. The primary causes tometry (Figure 5). Apoptosis was significantly of MM-related death are tumor relapse and greater in bortezomib- and DATS-treated SP drug resistance [4]. These effects are associ- cells compared to that in DMSO-treated cells ated with the existence of SP cells, which have (P<0.05). The results also indicated that apop- features similar to those of tumor stem cells tosis was not significantly different between [5, 6]. The results of this study indicated that cells treated with bortezomib and cells treated the proportion of SP cells was 2.08±0.438% with DATS (P>0.05). and 1.26±0.352% among RPMI-8226 and NCI-

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